Plasma membrane nucleoside transporters are important in transporting nucleoside drugs into their target cells where they are metabolized and become cytotoxic. Nucleoside drugs are clinically used in the treatment of cancer (eg. Ara C in leukemia) and viral diseases (eg AZT in AIDS). Multiple nucleoside transporters have been cloned and are divided into Na-dependent Concentrative Nucleoside Transporters (CNTs) and Na-independent Equilibrative Nucleoside Transporters (ENTs). There is no homology between CNTs and ENTs. Our laboratory has cloned a new member of ENT gene family, human ENT3, and a new member of CNT gene family, human CNT3. Transient expression of hCNT3 in Cos7L cells confirms that hCNT3 is a broadly selective nucleoside transporter. For hENT3, immunolocalization studies show that hENT3 is a Golgi nucleoside transporter. Expression of hENT3 in PS120 fibroblasts containing functional plasma membrane nucleoside transporters, confers the cells resistant to the cytotoxicity of nucleoside drugs but not to the cytotoxicity of nucleobase drugs. Thus, we hypothesize that overexpression of hENT3 accounts for drug resistance, while overexpression of plasma membrane nucleoside transporter (hENT1, hENT2 or hCNT1-3) accounts for increase in drug sensitivity. In this application, we propose to characterize the kinetic and pharmacological properties of hENT3 (Aim1) and hCNT3 (Aim2). Strategic approaches include cell biology (immunolocalization and production of nucleoside transporter antibodies), biochemistry (membrane fractionation and reconstitution), molecular biology (targeting tag and site-directed mutagenesis), physiology (characterization of endogenous nucleoside transport systems) and pharmacology (drug resistance and nucleoside drug transport). A long-term goal of our studies is to use the knowledge of nucleoside transport at the molecular level to facilitate the design of nucleoside analogue drugs for the treatment of various viral, parasitic, and neoplastic diseases and as cardiovascular protective agents. The identification of hENT3 as a Golgi nucleoside transporter opens a new venue for studying molecular mechanisms of drug resistance.